JPH0553140B2 - - Google Patents

Abstract

Aqueous suspensions of fibrillar atelopeptide collagen and osteogenic factor are effective injectable preparations for the repair of bone defects. Processes for the production of such preparations are described.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention belongs to the fields of protein chemistry and osteogenesis. Injectable preparations.

BACKGROUND OF THE INVENTION Historically, methods for bone repair have advanced and now include techniques to replace lost bone material and aid the bone cells' ability to perform proper repair. Various compositions are now available. Previous researchers have recognized that protein factors present within bone tissue are responsible for the maturation of bone progenitor cells into bone tissue. Not all of these factors have been identified so far, nor have their characteristics been completely clarified. However, based on the presence or absence of these factors, preparations can be classified as "conducting" and "inducible" preparations, depending on whether or not these preparations themselves supply these factors. It is now possible to classify

"Conductive" preparations do not contain osteogenic factor (OF). They repair bone in a "passive" manner by providing a matrix for new growth. There are many types of these compositions. Of particular relevance to the preparations of the invention are bone repair compositions consisting primarily of certain collagens, the organic structural materials of bone. It is believed that the collagen matrix can provide the desired structure for new bone ingrowth into the defect. In fact, when this matrix comes into contact with mature bone cells, it can penetrate into them and create a mineral network to complete bone growth. Thus, living tissue in contact with this matrix provides all the requirements for OF. In fact, in recent years, various cases have been reported in which collagen itself or collagen mixed with hydroxyapatite is used for this purpose.

In this way, bone repair can be achieved by not only passively leading to the invasive growth of new bone tissue and subsequent mineralization, but also by actively inducing osteogenic cells to transform into osteocytes. . In other words, it goes without saying that it is desirable to create a bone repair matrix that has an "inductive" repair action. The process of bone formation in vivo is complex and only partially understood. but,
This is thought to involve the intermediate formation of chondrocytes or cells capable of making cartilage, which are replaced by cells capable of effecting mineralization. Recently, research has been carried out on materials that can bring about such differentiation.

It is certain that bone itself contains one or more factors involved in its formation process. Therefore, efforts are being made to clarify what factors bring about this effect. Urist
No. 4,294,753 and No. 4,455,256 disclose that "bone morphogenetic proteins" can be extracted from bone demineralized using urea or guanidine hydrochloride.
(BMP) was extracted and reprecipitated. Urist then uses ion exchange to purify this crude protein mixture, which is not absorbed by carboxymethylcellulose resin (CMC) at a pH of 4.8.
reported that activity occurred (Urist, MR,
Clin Orthop Rel Res (1982) 162:219). Urist
Report of Science (1983) 220:680-685 and
Proc Natl Acad Science (USA) (1984) 81:
371-375) describe BMPs with molecular weights of 17,500 Daltons and 18,500 Daltons.
According to Urist's most recent European Patent Application Publication No. 0212474, by limited proteolysis of BMPs,
A BMP fraction of 4000 to 7000 daltons is obtained.

Seydin and Thomas, US Pat. No. 4,434,094, describes extraction with chaotropic reagents, fractionation on anion and cation exchange columns, and PH
In Section 4.8, we report on the partial purification of bone protein that promotes bone formation by restoring its activity from the fraction adsorbed to CMC. This new protein fraction is called "bone morphogenetic factor" (OF) and has a molecular weight of approximately 30,000
It has the characteristic of being less than Dalton.

Co-owned US Pat. No. 4,774,322 describes two proteins purified to homogeneity using a purification method partially similar to that disclosed in US Pat. No. 4,434,094. These two proteins were purified by CMC in a NaCl concentration gradient of approximately 150-200mM.
eluted from. These two proteins were originally called cartilage inductive factor (CIF) A and CIF B. CIF A is then a previously identified protein, transforming growth factor beta (TGF
-b). CIF B was also found to be a new species of TGF-b, and is now known as TGF-b2. Both of these themselves have no osteogenic activity in vivo.

Co-owned U.S. Patent No. 4,627,982 describes TGF-b
and within the CMC-bound fraction of U.S. Pat. No. 4,434,094, which is eluted in the lower NaCl gradient portion (i.e., lower than about 150 mM NaCl) than the NaCl gradient in which the major portion of TGF-b2 is eluted. , a partially purified osteoinductive factor.

As a result of the availability of these specific inducible repair factors in unpurified or pure form,
Attempts were made to formulate preparations for implantation into bone defects. US Pat. No. 4,440,750 discloses an osteogenic composition comprising reconstituted collagen fibers and demineralized bone powder. This patent suggests a method of formulating the mixture in injectable form. However, it is impractical, if not impossible, to deliver a practical concentration of the mixture by syringe because the bone powder particles clog the needle. U.S. Pat. No. 4,394,370 (Jefferies) describes a composition containing unspecified collagen and demineralized bone particles or dissolved bone morphogenetic protein (possibly Urist's bone morphogenetic protein). Disclosed. The Jefferies material is described as a sponge suitable for implantation into bone defects. No method has been shown to evaluate the inducible effects of this implant. US Patent No. 4563350
describes a partially purified graft composition by adsorption and desorption from CMC or by treatment of the low molecular weight fraction of the extract with an anion exchange resin at pH 7 and recovery of the unbound fraction. ing. These factors are administered with a collagen carrier containing at least 5% by weight of collagen in non-fibrous form. These formulations were constructed as solid bone grafts.

US Patent No. 4,687,763 describes a solid implant that promotes bone growth prepared by precipitating demineralized bone extract onto a flexible collagen support. This preparation is
This is done by adding a solvent for the extractant solvent to the extract solution.

None of the above compositions are practical injectable inductive preparations. An advantage of such injectable preparations is that surgical procedures commonly required when using non-injectable inducible preparations can be avoided. The present invention relates to injectable preparations containing fibrillar collagen and at least partially purified OF.

SUMMARY OF THE INVENTION The present invention is directed to a novel injectable preparation that can be used for bone repair in humans and other mammals. In its simplest form, this preparation contains an aqueous suspension of fibrillar atelopeptide collagen and at least partially purified osteogenic factor. Other active ingredients can be added to this preparation, such as bone growth promoters (eg TGF-b) and chemotactic factors that can promote bone formation. These added substances must not impair the injectability of the composition.

Also forming part of the invention is a method of inducing bone growth at a site in a living mammalian individual by injecting the preparation into the site.

Methods of making the preparations of the preferred embodiments are also yet another aspect of the invention.

The method includes the following steps: (a) preparing an acidic aqueous solution of atelopeptide collagen and bone morphogenetic factors; (b) increasing the pH of the solution to increase the pH of atelopeptide collagen and bone formation factor; and (c) optionally adjusting the concentration of the coprecipitate in the resulting suspension to about 5 to about 65 mg/ml based on the atelopeptide collagen. .

Structure of the Invention A. Preparation of the Collagen Component of the Solution A suitable collagen for preparing the injectable composition of the present invention is an acidic aqueous solution of atelopeptide collagen. Preparation of this solution is well known in the art and is available from VITROGEN 100 Collagen Solution (CIS) (Collagen Corporation, Palo Alto;
Preparations such as CA) are commercially available. Other collagens suitable for making the injectable preparations of the invention include reconstituted fibrillar collagens, which use CIS as a starting material or
Zyderm Collagen Implant (ZCI)
Corporation, Palo Alto, Calif.). but,
The method for preparing the collagen component is not particularly limited, and a method for obtaining a suitable material is outlined below. The obtained collagen has a high degree of purification,
Any collagen component of mammalian origin can be used as long as it does not contain atelopeptides which are believed to be at least partially responsible for immunogenicity. Solubilization creates a purified form of collagen that reduces immunogenicity.

In a suitable manufacturing method, a mammalian leather preparation, preferably bovine leather, is softened by soaking it in a mild acid and then scraping off the hair, epidermis, and fat. The tweezed leather is again immersed in a weak acid, ground or chopped to produce a finely divided preparation, which is dispersed in an aqueous medium and further treated with a proteolytic enzyme other than collagenase, preferably a It is solubilized under non-denaturing conditions by being degraded by enzymes that are also active at PH. As the acid solution, for example, a diluted acid solution of HCl or a carboxylic acid (eg, acetic acid, malonic acid, lactic acid, etc.) is used at low temperature, usually at a pH of 1.5 to 5 depending on the enzyme used. A preferred method is to incubate the minced tissue in HCl at approximately pH 20°C.
In step 2, it is dispersed at a concentration of 1 to 5 g/. After dispersing the tissue, enzymes are added and the mixture is incubated to enzymatically degrade telopeptides and other soluble components of the tissue. Enzymes suitable for decomposing telopeptides that do not attack the triple helix portion of collagen include:
Pepsin, papain, and trypsin (preferably trypsin) are included, with concentrations of these enzymes ranging from 0.1 to 10, based on the collagen content of the tissue.
% by weight. The incubation period is about two days to two weeks, and the solubilization process can be monitored by measuring the viscosity of the solution. Once this viscosity reaches a substantially steady level, solubilization is complete and the enzyme is inactivated and removed.

After denaturing the enzyme, the denatured enzyme and the degraded portion of the tissue are removed using various methods (e.g., dialysis,
The solution is removed by sedimentation, filtration), or a combination of these methods.
Soluble components, including collagen, are separated from the sedimented or filtered solids, concentrated, optionally fractionated by ion-exchange chromatography, and then further concentrated to produce a substantially pure atelopeptide collagen solution. Prepare. Typical concentration levels of collagen (CIS) in solution are 3-25mg/ml
It is. This CIS is carried out at low temperatures, preferably around 10-25
C., and can be reconstituted into fibrils by neutralizing the solution, preferably under hypotonic conditions relative to physiological ionic strength. The neutralizing solution can be added directly or preferably by dialysis of the solubilized collagen. An ionic strength of about 0.03-0.1M is used. The PH can be raised to a level where collagen in solution reaggregates into fibrils by adding a suitable base or buffer (eg, disodium phosphate or sodium hydroxide). Under these conditions fibrils are formed when the PH is in the range of about 5-10, but preferably the final PH is in the range of 5-8. The duration of fibril formation is typically
It ranges from approximately 30 minutes to 18 hours.

B. Preparation of the osteogenic factor component in solution A partially pure demineralized bone extract containing osteogenic factor or a purified form of this factor can be used. For example, U.S. Pat.
Partially purified factors described in US Pat. No. 4,627,982 can be used. In this regard, the term "at least partially pure" refers to demineralized bone extracts of non-fibrous proteins, preferably at least as described in US Pat. No. 4,627,982.
means purified to the level specified in the item. Partially purified factor is 0.01-0.1M
Further purification can be achieved by precipitation in a phosphate buffer of pH 6-8 by addition of sodium phosphate. This precipitate is then redissolved in a nonionic chaotropic reagent and chromatographically analyzed by HPLC using a hydroxyapatite column. In any case, the method for obtaining this factor from bone is as follows.

The bones are first cleaned and ground by mechanical methods or friction, washed, for example with a dilute aqueous acid solution, preferably at low temperature, and extracted with a lipophilic solvent (for example, ether or ethyl acetate). Degrease. Next, this bone
It is usually demineralized by extraction with stronger acids to remove the various forms of calcium phosphate. These techniques are well known in the art and are disclosed, for example, in US Pat. No. 4,434,094. The resulting preparation, ie demineralized bone, serves as the starting material for the bone morphogenetic protein preparations of the invention.

The first extraction is aimed at removing non-fibrous (eg, non-collagen) proteins from the demineralized bone. This includes guanidine hydrochloride (at least about 4 moles), urea (8 moles) and salts,
or sodium dodecyl sulfate (at least about 1
% by volume) or other chaotropic reagents well known in the art (Termine et al., J Biol Chem (1980) 255:9760).
−0722; and Sajera and Hascall, j Biol
Chem (1969) 244:77-87 and 2384-2396). Extraction is preferably performed at low temperatures in the presence of protease inhibitors to reduce the possibility that the extracted proteins will be degraded or denatured. Examples of protease inhibitors that can be used include phenylmethylsulfonyl fluoride (PMSF), [sodium azide], N-ethylmaleimide (NEM), benzamidine, and 6-aminohexanoic acid. The PH of the medium is determined depending on the selected extractant. The time required for extraction is generally about 4 hours to about 1 day.

After extraction, the extractant can be removed by suitable means, such as first concentration by ultrafiltration, if necessary, followed by dialysis against water. Salts can also be removed by controlled electrophoresis or molecular sieving, or other means well known in the art. It is preferred to maintain low temperatures during this step to minimize protein denaturation. Also, the extractant chaotropic reagent does not necessarily have to be removed; the solution can simply be concentrated, for example by ultrafiltration.

The extract dissolved or redissolved in a chaotropic reagent is subjected to gel filtration, and the molecular weight of
Obtain fractions in the range of 35,000 Daltons. Gel size classification is performed using standard methods. This was carried out using a Sephacryl S-200 column at room temperature (10
It is preferable to carry out the reaction at a temperature of 25°C to 25°C.

The sized fractions are then converted to CMC at about PH4.5-5.2 (preferably about PH4.8) in the presence of a non-ionic chaotropic reagent (e.g. 6M urea).
The sample is subjected to ion-exchange chromatography using Preferred are cellulose cation exchangers, although other cation exchangers (including those derived from polyacrylamide and cross-linked dextran) may also be used. Of course, in any ion exchange method, the solution must be free of competing ions before being applied to the column. The above factors are adsorbed onto the column and about 10 to about
It is eluted with an increasing salt concentration gradient in the range of 150mM. This fraction is conveniently referred to as "CMB-1." CMB-1 can be obtained by dialysis or
Desalted using a C ₁₈ reverse phase column.

C. Preparation of Compositions of the Invention Compositions of the invention can be prepared by co-precipitating CIS with OF or by simply mixing reconstituted fibrillar collagen with OF.

1 Co-precipitation A preferred method of mixing fibrillar collagen and OF is by coprecipitation. Alternatively, mixing the two components using a mechanical mixer or homogenizer is less desirable. A preferable coprecipitation method is the following method. First add OF to an acid (e.g.
0.01N HC1, PH2.0), approximately 3μg/ml to 3mg/ml
Solubilize at a range of concentrations. The CIF/OF mixture is
Co-precipitation is carried out by increasing the pH of the solution to about 6.5-8.5, preferably about 7-7.5. This is done by adding a base, preferably 0.2M phosphate buffer (PH11.2). The obtained precipitate is
Centrifuge and resuspend at desired concentration in injectable medium. The concentration of the coprecipitate in the suspension is usually 5 to 65 mg/ml, preferably 10 to 25 mg/ml.
ml range. at least partially purified
The weight ratio of collagen to OF is usually 5:
The ratio is 1 to 300:1.

2. Mixing Suitable methods for preparing injectable mixtures of collagen and OF vary as the concentration of one dictates the acceptable range of concentrations of the other. Therefore, it is preferable to consider the necessary parameters of the final composition. To enable administration by injection, the concentration of reconstituted fibrillar collagen should be between 5 and 65 mg/ml.
ml, preferably 10-20 mg/ml. Reconstituted collagen suspension with higher collagen concentration is
It can be used when mixed with an OF solvent to lower the collagen concentration. The weight ratio of collagen to partially pure OF in the mixture is the same as above.

As indicated above, inert or biologically active substances can be further added to this preparation. These additive materials must not impair the injectability of the composition.

E Dosage Forms The compositions of the invention are prepared as injectables. These compositions are administered to the defect by conventional injection techniques well known to physicians. In this regard, these compositions are conveniently loaded into a suitable sterile administration syringe. These compositions should be stored at low temperatures (e.g., about 4° C.) prior to use and administered in amounts commensurate with the size of the defect and the composition's intended role in treating the defect. .

(Examples) The following examples further illustrate the present invention, but are not intended to limit the invention.

Example 1 A partially purified CMB-1 fraction was prepared from bovine bone powder according to US Pat. No. 4,563,350. Briefly, a bovine metatarsal with 0.5N
Demineralized in HC1 and dissociatively extracted with 4M guanidine hydrochloride (GU.HC1). GU.HC1
After concentration, the extract was filtered through a gel filtration column (S-200).
A low molecular weight protein (F35 K Dalton) was obtained. This low molecular weight protein was further concentrated, desalted with a GH-25 column, and applied to a cation exchange column (CM-52) to obtain a CMB-1 fraction. CMB-1 protein was finally desalted by applying it to a reverse phase HPLC column (C18) and directly lyophilized.

CMB-1 protein was coprecipitated with collagen as follows. All steps were performed in a laminar flow hood. Freeze-dried CMB-1,
It was dissolved in 0.01NHC1 (PH2.0) at a protein concentration of approximately 2.5 mg/ml. This solution was sterile filtered through a 0.22 mm filter and treated with VITROGEN 100 CIS (3 mg/ml).
mixed with. The mixture was stirred for approximately 5 minutes to thoroughly mix. For co-precipitation of CMB-1 protein and collagen, 1 part of 0.2M is added to 9 parts of solution.
This was done by adding phosphate buffer (PH11.2). Stir this mixture for about 5 minutes and leave it for about 5 minutes.
It was left in the hood for 18 hours. The resulting precipitate, a mixture of CMB-1 protein and fibrillar collagen (FC), was heated at 12000 RPM for 20
Centrifuged for a minute and separated from the supernatant. This supernatant was saved for protein measurement. The precipitate was collected and its wet weight was measured. From the measurement results so far, approximately 85% of the total collagen protein is CIS
It is expected that it will precipitate in the form of FC. The FC concentration was determined using the above supernatant and 1.3M NaCl, 0.02M sodium phosphate buffer (PH7.2) as the medium for precipitate dilution in 0.13M NaCl, 0.02M sodium phosphate buffer. The concentration was adjusted to 15 mg/ml. The FC/CMB-1 complex was thoroughly mixed by homogenizing between syringes. The homogenized sample was then loaded into a 1 c.c. syringe and stored frozen (4° C.) until use.

The ability of the FC/CMB-1 coprecipitate to induce mammalian cartilage tissue formation was confirmed as follows.
Sprague-Dawley rat (34-40 days old male)
0.2 cc of FC/CMB-1 mixture was injected subcutaneously into one side of the abdominal thorax. A sample of FC alone without CMB-1 was also injected as a negative control. The implants were removed after 7 and 14 days, and the state of cartilage and bone formation was examined biochemically and histologically.

Table 1 below summarizes the results of the histological examination.

Table 1 Group 7 days post-transplant 14 days post-transplant Cartilage Bone Cartilage Bone FC/OF 4+ 2+ 2+ 4+ FC control 0 0 0 0 Histologically, the formulation containing the coprecipitate was
It was shown that cartilage formation was significantly induced within 7 days after transplantation. Also, at this time there were signs of early bone formation around the implant. Supporting this observation, alkaline phosphatase (AP) activity is increased in these implants (approximately 35 units AP/g of implant). No biochemical activity was observed in the control test group injected with FC-only samples. 14 days after injection,
All of the FC/CMB-1 implants showed increased bone formation and bone marrow was almost discernible. Cartilage activation was absent or at very low levels in the center of the implant. Histologically, there were no signs of inflammation due to the implant. This indicates that the implant is compatible with the host tissue.

Example 2 The coprecipitate was the same as Example 1, except that a more highly purified bone morphogenetic protein (purified by ConA affinity chromatography, as described above) was used instead of CMB-1 protein. It was prepared and tested in the same manner.

The results obtained were similar to those obtained in Example 1, and extremely high osteoinductive activity was observed in the implant containing the coprecipitate.

Example 3 65mg/ml Zyderm Collagen Implant (ZCI)
The material was diluted with 20mM phosphate buffer (PH7.4) containing 0.13M NaCl to a concentration of 35mg/ml. The diluted collagen was prepared in Example 1.
Thoroughly mix with CMB-1 protein of 1.2 mg/ml.
The final concentration of collagen was 30 mg/ml for CMB-1 protein. The proportion of OF in this composition is approximately 3.8%. This mixture was then loaded into a 1 c.c. syringe and stored at 4°C until injection.

ZCI/CMB induces cartilage tissue formation in mammals
The ability of the -1 mixture was confirmed as follows. Four young male Sprague-Dawley rats were injected subcutaneously into one side of the abdominal thorax with 0.5 cc of ZCI/CMB-1. Reconstituted and Demineralized Bone (R-
DBP), and ZCI alone. Control injections of ZCI were performed using 0.5 cc of ZCI, and control sample R-
DBP (40 mg) was surgically implanted.

The implants were removed after 14 and 28 days, and the state of cartilage and bone formation was examined biochemically and histologically. Histological evaluation was performed after fixation, sectioning, and staining by standard procedures. Biochemical determination was performed by analyzing cartilage proteoglycan content and specific activity of alkaline phosphatase.

Histological criteria were chondrogenicity, osteogenesis, ductalization, and fibroblast invasion. Based on these criteria, the collagen control injection showed no activity except for slight ductalization and fibroblast invasion after 28 days;
After 14 days, these were no longer observed. ZCI／
Both the CMB-1 and R-DBP compositions showed activity on all four of these criteria, except that the R-DBP composition had higher ZCI/
It showed slightly equal and greater activity than the CMB-1 composition.

The proteoglycan activity analyzed after 14 days was
When only ZCI is used, it is not recognized at all, and ZCI/
When using CMB-1, 550 mg per gram of wet tissue
When using C-PG, R-DBP, 1 g of wet tissue
There was 1300 mg of C-PG per serving.

Alkaline phosphatase activity was analyzed after 14 and 28 days; again, virtually no alkaline phosphatase activity was observed in the explants from animals injected with ZCI alone. Implants from rats injected with ZCI/CMB-1 had 8.5 U/g of wet tissue after 14 days and 14.5 U/g of AP after 28 days. R
The alkaline phosphatase activity of implants removed from rats injected with -DBP was also 12 U/g after 14 days and 20 U/g after 28 days.

From the above, injectable mixed ZCI/CMB
It can be concluded that the -1 composition may have comparable osteogenic activity as the R-DBP preparation.

Modifications of the above-described modes of carrying out the invention that are obvious to those skilled in the art of protein chemistry and osteogenesis are also within the scope of the permitted claims.

SUMMARY OF THE INVENTION An aqueous suspension of fibrillar atelopeptide collagen and bone morphogenetic factors is an effective injectable preparation for repairing bone defects.

Claims (1)

Claims: 1. An injectable bone growth inducing composition comprising an aqueous suspension of fibrillar atelopeptide collagen and at least partially purified osteogenic factor. 2. The composition of claim 1, wherein the concentration of said collagen ranges from about 5 to about 65 mg/ml. 3. The composition of claim 1, wherein the fibrillar atelopeptide collagen and the at least partially purified osteogenic factor are in the form of a coprecipitate. 4. The composition of claim 2, wherein the weight ratio of collagen to osteogenic factor of the coprecipitate ranges from about 5:1 to 300:1. 5. The composition of claim 4, wherein the osteogenic factor is co-precipitated from a demineralized acidic bone extract. 6. A method for producing the composition according to claim 4, comprising: (a) preparing an acidic aqueous solution of atelopeptide collagen and bone morphogenetic factor; (b) increasing the pH of the solution; co-precipitating the atelopeptide collagen and the osteogenic factor; and (c) optionally adjusting the concentration of the coprecipitate in the resulting suspension to about 5 to 5, based on the atelopeptide collagen. A manufacturing method comprising: adjusting the concentration to about 65 mg/ml. 7. The method of claim 6, wherein the osteogenic factor is present as a demineralized bone extract. 8 The weight ratio of atelopeptide collagen to demineralized bone extract in the solution is about 5:
The manufacturing method according to claim 7, wherein the ratio is 1 to 300:1. 9. The method of claim 6, wherein the pH of the solution is increased from about 6.5 to about 8.5.